Initiator tRNAs from prokaryotes and eukaryotes possess certain characteristic structural features that distinguish them from the elongator tRNAs. In eukaryotes, the first step in translation initiation involves the formation of a highly specific ternary complex between initiator Met-tRNA and elF2.GTP. Formation of the ternary complex is a rate-limiting step in translation; it is also the step of global translational regulation. In spite of the importance of the ternary complex, there is very little information on the determinants on the initiator tRNA required for binding to eIF2. This proposal aims to investigate: (1) the importance of the amino acid attached to the initiator tRNA in binding to elF2, (2) the sequence and/or the structural elements in the initiator tRNA required for binding to elF2, and (3) the topology of initiator Met-tRNA-elF2.GTP interaction. Specific mutant initiator tRNAs will be made that will lack one or more of the structural features unique to eukaryotic initiator tRNAs. The effect of these alterations on initiation of protein synthesis and on binding to elF2 will be investigated using both mammalian cells and experiments in vitro.